Laboratory of Protein Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussel, Belgium.
Fish Shellfish Immunol. 2010 May-Jun;28(5-6):743-53. doi: 10.1016/j.fsi.2010.01.007. Epub 2010 Jan 28.
From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 microg SAP and 56 microg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1-2 mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca(2+) ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.
从 Pangasianodon hypophthalmus 的血清中，通过琼脂糖和磷酸胆碱琼脂糖的亲和层析分离出两种蛋白质。它们在亲和基质上的结合严重依赖于 Ca2+ 离子的存在。N 端测序和内部胰蛋白酶肽的测序将蛋白质鉴定为五聚蛋白，根据其结合特性，它们被鉴定为 SAP（血清淀粉样蛋白 P 成分）和 CRP（C 反应蛋白）。每毫升血清中，SAP 被纯化至 36 微克，CRP 被纯化至 56 微克。在凝胶过滤中，SAP 和 CRP 均作为分别由 24 kDa 和 28 kDa 亚基组成的三聚体洗脱。两种蛋白质均不含链间二硫键。SAP 和 CRP 均为糖基化的，可凝集兔红细胞和病原菌爱德华氏菌和嗜水气单胞菌，但不能凝集微球菌和大肠杆菌。SAP 和 CRP 的血凝作用分别被半乳糖（MIC = 1 mM）和磷酸胆碱（MIC = 1-2 mM）抑制。圆二色性研究表明，反平行的 β-折叠片是主要的二级结构。通过 EDTA 去除 Ca（2+）离子后，在近紫外区通过 CD 光谱观察到轻微的结构变化。免疫扩散表明，P. hypophthalmus SAP 和 CRP 不发生交叉反应。
